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Journal of Chemical and Pharmaceutical Research, 2014, 6(8):156-161

ISSN : 0975-7384 Research Article CODEN(USA) : JCPRC5

Evaluation of antibacterial activity of extracts from aventis

Abdul-Malik Abudunia*1,2,4 , M’Hammed Ansar 2, Jamal Taoufik 2, Youssef Ramli 3, El Mokhtar Essassi 3, Azeddine Ibrahimi 1 and Khadija Khedid 4

1Biotechnology Laboratory (Med-Biotech), Faculty of Medicine and Pharmacy, Mohammed V University-Souissi, Rabat, Morocco 2Department of Medicinal Chemistry Laboratory, Faculty of Medicine and Pharmacy, Mohammed V University- Rabat, Morocco 3Department of Heterocyclic Organic Chemistry Laboratory, Faculty of Sciences, Mohammed V University-Agdal, Rabat, Morocco 4Department of Bacteriology, National Institute of Health (INH), Avenue, Ibn Batouta -Agdal, Rabat, Morocco ______

ABSTRACT

Calendula arvensis (C.A.) is one of the herbs used in traditional medicine in Morocco , Due to its essential oil it is recognized by its smell and warmth characteristic .This work aims to evaluate the antibacterial activity of C.A. extracts , Hexanolic and methanolic extracts were obtained by the method of soxhlet , while aqueous extract was obtained by maceration in cold water. Antibacterial activity screening was conducted by the well diffusion method according to the clinical and laboratory standard institute guidelines using 6 Gram positive and 7 Gram negative bacteria (sensitive and multidrug-resistant) .The results indicated that , the methanolic extracts of C.A. flowers generally showed high inhibitory activity against Gram-positive and Gram negative bacteria except Acinetobacter baumanii , Proteus mirabilis and Listeria monocytogenes, The C.A. flowers hexanolic extract inhibited all bacteria of Gram-positive and Gram negative bacteria except, Staphylococcus aureus MRSA, Streptococcus agalactiae and Acinetobacter baumanii , There were no inhibitory effects of the aqueous extracts against all tested bacterial strains except Rhodococcuss equi and Morganella morganii, MICs values of methanolic extracts were between 12.5-25 µg/ml . while MICs of values of hexanolic extracts were between 6.25- 12.5 µg/ml . In conclusion , the obtained results show an antibacterial potency of organic extracts of C.A. flowers. C.A. flowers Hexanolic extracts were bactericidal for most bacteria while methanolic extracts were bacteriostatic .

Key words: , antibacterial activity, MICs, MBCs, medicinal . ______

INTRODUCTION

In Morrocco , as elsewhere in the world, are widely used in the treatment of various diseases . The varied climate and heterogeneous ecologic condition in Morocco have favoured the proliferation of more than 42,000 species of plants, divided into 150 families and 940 genuses [1]. Drugs derived from natural sources play a significant role in the prevention and treatment of human diseases. In many developing countries, traditional medicine is one of the primary healthcare systems [2,3]. Herbs are widely exploited in the traditional medicine and their curative potentials are well documented [ 4]. Therefore, in the traditional medicine prevention and treatments of several humans diseases is succefully controlled by using aromatics medicinal plants . Recently, in pharmaceutical industry, about 61% of new drugs developed between 1981 and 2002 derived from natural sources and they have been very successful, especially in the areas of infectious disease and cancer [ 5].Natural products of higher plants may give a new source of antimicrobial agents with possibly novel mechanisms of action [ 6,7]. The effects of plant extracts on bacteria have been studied by a very large number of researchers in different parts of the world [ 8] . Plants are rich in a wide variety of secondary metabolites such as tannins, terpenoids, alkaloids, flavonoids,

156 Abdul-Malik Abudunia et al J. Chem. Pharm. Res., 2014, 6(8):156-161 ______glycosides, etc., which have been found in vitro to have antimicrobial properties [ 9,10 ]. Herbal medicines have been known to man for centuries. Therapeutic efficacy of many indigenous plants for several disorders has been described by practitioners of traditional medicine [ 11 ]. Antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world. The World Health Organization estimates that plant extracts or their active constituents are used as folk medicine in traditional therapies of 80% of the world's population [ 12 ]. The harmful microorganisms can be controlled with drugs and these results in the emergence of multiple drug-resistant bacteria and it has created alarming clinical situations in the treatment of infections. The pharmacological industries have produced a number of new antibiotics resistance to these drugs by microorganisms has increased. In general, bacteria have the genetic ability to transmit and acquire resistance to synthetic drugs which are utilized as therapeutic agents [ 13 ].

Calendula L . ( ), usually known as “marigold ”, is a reputed medicinal plant with ornamental properties. The yellow or -colored flowers are used as food dye, spice, and tea as well as tincture, ointment or cosmetic cream. Although the genus Calendula is usually indigenous to the southern European region including Italy, Malta, Greece, Turkey, Portugal, and Spain [14], it is nowadays cultivated in many temperate regions of the world depending on its commercial value. Since is grown in northern parts of Africa, it is also named as “African marigold ” [15]. Calendula is a relatively small genus (about 12–20 species native to the Macaronesian and Mediterranean regions are currently recognized) that belongs to the Asteraceae family (tribe: Calendula ) [16] . Calendula arvensis C.A. the common name of which is field marigold, is an annual herbaceous species (10–100 cm tall) that is widespread in central and southern Europe, northern Africa, southwestern Asia and the Macaronesian region [17] . The leaves, which contain tector and secretory trichomes, are lance shaped. The consists of a single flower head with yellow or orange capitula (blossoms are produced year round, but flowering peaks between March and July). From a taxonomic viewpoint, C.A. is a complex species because of polymorphism in the size of the stems (5–60 cm), width of the leaves (5–20 mm), color of the flowers (bright yellow to yellow-orange) and the size and shape of the three types of achene (rostrate , cymbiform and annular) [18]. Thus, various subspecies have been ascribed to C.A. in various Mediterranean areas [16]. Numerous investigations have proved that extracts of the aerial parts of C.A. are rich in sesquiterpene glycosides (based on their alloaromadendrane, eudesmane and cubebane skeletons) [19]. Furthermore, phytochemical studies have shown that C.A. contains various families of compounds such as the phenolic acids and the flavonoids. To our knowledge, no data are available on the chemical composition of essential oil obtained from C.A. Several investigations on the volatile constituents of Calendula officinalis (also known as the pot marigold) have been conducted in which volatiles were extracted by hydrodistillation [20]. Calendula officinalis oils are generally characterized by a high content of sesquiterpenes with cadinane or muurolane skeletons. It should be noted that this species is cultivated worldwide for ornamental and medicinal purposes. It is also used in the manufacture of cosmetics, perfumes and pharmaceuticals and in the food industry to add color or flavor. There is only one report on the chemical composition of the essential oils of other Calendula species, viz., a report on the essential oil composition of the flowers of Calendula micrantha plants in Egypt [21] . The main components were aromadendrene, a-gurjunene, a- pinene, benzyl formate, (E)-b-caryophyllene and guaiol [22].

The aim of this study is to evaluate the antibacterial activity of C.A. flowers of methanolic extracts, hexanolic and aqueous extracts, and to, therefore, determine the scientific basis for its use in traditional medicine in the treatment of infection diseases. To the best of our knowledge, this is the first study to do so against a wide range of bacteria.

EXPERIMENTAL SECTION

Plant Materials: Flowers of Calendula arvensis were collected based on ethno pharmacological information from the villages around the region Rabat-Temara -Sidi El-Abed, during the month of March 2011 starting around 7 AM each morning. All agreements were obtained from the authorities with respect to the United Nations Convention of Biodiversity and with assistance of traditional medical practitioner. The plant was identified by botanist From the Moroccan Scientific Institute (Rabat). Avoucher specimen (N°RAB 78161) was deposited at the Herbarium of the Botany Department at the same institution.

Preparation of the extracts : Aqueous Extract (AE) : The powdered of C.A. flowers extracts (200g)were extracted with boiling water (200 ml) for 30 min . The decoction was filtered and then freeze-dried (aqueous extract) [23].

Organic Extracts (OE) : Hexanolic extract (H.E.) and Methanolic extract (M.E.) were obtained by using the Soxhlet extraction of 200 g of C.A. flowers of C.A. in about 700 ml of solvents for 24 h. The filtrated solvents were evaporated in vacuo until dryness using rotator evaporator. These extracts were concentrated to dryness and the residues were kept at 4°C [23].

157 Abdul-Malik Abudunia et al J. Chem. Pharm. Res., 2014, 6(8):156-161 ______

Antibacterial activity : Preparation of test samples : The hexane and methanol extracts were dissolved in dimethylsulfoxide (DMSO) solvent . As a precaution not to miss trace amount of antimicrobial agent for preliminary screening, a relatively high concentration of 200 mg/ ml of each fractions were prepared for bioassay.

Bacterial strains and culture conditions : The extracts of C.A. flowers were individually tested against 6 Gram positive 7 Gram negative bacteria microorganisms including : Rhodococcus equi , Staphylococcus aureus MDRSA, Staphylococcus aureus MLSA, Streptococcus agalactiae ,Enterococcus faecalis , Listeria monocytogenes ,Pseudomonas aeruginosa , Pseudomonas cepacia, Pseudomonas aeruginosa ATCC and Acinetobacter baumanii , Proteus rettegeri MDRPR, Proteus mirabilis , Morganella morganii . These strains were obtained from American Type Culture Collection (ATCC), USA ; Bacteriology Laboratory of Institute National of Health (INH) and Institute of Agronomy and Veterinary medicine (IAV), Morocco. All media used in this study, were manufactured by Oxoid (Basingstoke,Hampshire,England). All bacteria were stored in BHI (Brain and Heart Infusion broth) containing 30 % (v/v) glycerol (Sigma-Aldrich) at -20°C. Prior to use bacterial strains were first grown in Muller Hinton agar (MHA) at 37°C for 24h. An aspartate aminotransferase (AST) of ciprofloxacin, Clindamycin and Gentamicin were also determined in parallel experiments in order to control the sensitivity of the Standard test organisms ( Staphylococcus aureus ATCC 25923).

Antibacterial activity screening : The antibacterial activity of all extracts was evaluated by the agar well diffusion method according to clinical laboratory standards directives (CLSI, 2007). The cell suspension was adjusted with sterile saline solution to obtain turbidity comparable to that of a 0.5 McFarland standard (1.5 × 108 cells mL-1). Therefore, the microorganisms were spread on Muller Hinton agar by cotton swab. Wells of 6 mm diameter were punched into the agar medium and filled with 50 µml of plants extracts. The plates were incubated for 24 h at 37°C. Antibacterial activity was evaluated by measuring the zone of inhibition against the test organism.

Minimum Inhibitory Concentration (MICs) : Are defined as the lowest concentration of C.A. extracts that will inhibit the visible growth of a microorganism after overnight incubation , The determination of MICs of extracts of C.A. flowers against the target test microorganisms was performed by microtitration technique as recommended by CLSI guidelines. The 0.5 McFarland suspension of bacterial strain from 24h culture in Muller Hinton agar, was appropriate diluted in saline water solution (0.85%w/v) to obtain the final inoculums to 105 cfu/ml .

Minimum bactericidal Concentration (MBCs) :Are defined as the lowest concentration of C.A. extracts that will prevent the growth of an organism after subculture on to antibiotic-free media , Minimum bactericidal concentration was carried out by subculturing 50 µl aliquots from each well. Least concentration of extract showing no visible growth on subculturing was taken as MBC value.

RESULTS AND DISCUSSION

Extract yield : The yield from C.A. flowers obtained by N-Hexane , Methanol and distilled water were found to be respectively 10,2, 20,5 and 20,3/200g of dry flowers. The result of this experiment showed that the methanol and distilled water fraction, which are the most polar was obtained in the highest yield, while the hexane fraction has the least yield (see Table I). This is an indication that the methanol and water distilled fraction contains more components compared to hexane fractions.

Table 1: Yields (w/w, %) of the C.A. flowers extracts

Type of solvents Extract yield (w/w, %) N-Hexane 10.2 Methanol 20.5 distilled water 20.3

Antibacterial activity: For the evaluation of antibacterial activity of extracts of C.A. flowers , we used the well diffusion method .

The results of this study showed that methanolic and the hexanolic extracts have rates of inhibition zone ( IZ ) very important compared to that of aqueous extracts (Table 11) . The greatest inhibition zone was obtained with methanolic and hexanolic extracts with means of inhibition zone are 14.8 mm and 12.5 mm , and The total rate of inhibition % is 77 and 68% respectively ( Table 11). Furthermore, it can be seen from (Table 11 ), Absence of antibacterial activity of aqueous extracts of C.A. could be explained by low quantity of active ingredients compared to that of the organic extracts. These results are in agreement with the literature which is generally found that the lowest antibacterial activity is associated with the aqueous extracts, Similar results were obtained during a

158 Abdul-Malik Abudunia et al J. Chem. Pharm. Res., 2014, 6(8):156-161 ______

study on the extracts of dichloromethane, methanol and an aqueous extracts of 30 medicinal plants in Yemen [24].

Table II. Antibacterial activity of different C.A. extracts against clinical bacterial pathogens

Antibacterial activity of C.A. extracts in term of inhibition zone (IZ) Groups of in mm bacterial The bacterial Strain tested Methanol N-hexane Water Strain Extract Extract Extract Rhodococcuss equi 28 24 18 Staphylococcus aureus MRSA 16 0 0 Gram + Staphylococcus aureus MLDSA 14 16 0 Streptococcus agalactiae 16 0 0 Enterococcus faecalis 21 16 0 Listeria monocytogenes 0 14 0 The total of inhibition 95 70 18 The means diameter of inhibition % 15.8 11.7 3 Pseudomonas aeruginosa 14 8 0 Pseudomonas cepacia 14 8 0 Pseudomonas aeruginosa ATCC 21 16 0 Gram - Acinetobacter baumanii 0 0 0

Proteus rettegeri MDRPR 20 14 0

Proteus mirabilis 0 21 0

Morganella morganii 28 26 16 The total of inhibition 97 93 16 The means diameter of inhibition % 13.9 13.3 2.3 Gram +, Gram - The means total of diameter of inhibition % 14.8 % 12.5% 2.6% IZ: Inhibition zone, MLDSA: Multi-drug resistance S aureus, MDRPR: Multi-drug resistance Proteus rettegeri , MRSA: Methicillin-resistance S aureus , ATCC : American type culture cell.

Figure I. Antibacterial activity of different C.A. extracts against clinical bacterial pathogens

Antibacterial activity of different C.A. extracts against clinical bacterial pathogens

18.00 16.00

14.00

12.00 Gram + 10.00

Gram -

8.00 Mean

6.00 4.00

2.00

0.00 Water Extract N-hexane Extract Methanol Extract

Antibacterial activity of C.A. extracts in term of inhibition zone (IZ) in mm

Methanol and water are polar solvents capable of extracting the plant material such as alkaloids, flavonoids, glycosides, tannins, salts, sugars and amino acids, with nearly the same yield of extraction. while the hexane is a non polar solvent, this feature gives it the property of extract : quinones, carotenoids, sterols, fatty acids, hydrocarbon [25], The effects observed with extracts of C.A. may be due to:

° Flavonoids , which in addition to their virtues spasmolytic , anti-inflammatory and anthelmintic, possess

159 Abdul-Malik Abudunia et al J. Chem. Pharm. Res., 2014, 6(8):156-161 ______antimicrobial activity. ° Flavonoids, which are present in extracts of C.A. could be the cause of its effectiveness as antimicrobial substances. This property is due to inhibition of bacterial enzymes, due to the reaction of addition their amine or thiol group. [26]. ° Carotenoids are molecules fungicidally power. Was able to demonstrate their antifungal activity against dermatophytes and Candida albicans as well as antibacterial action against certain Gram + and Gram-[26].

Table 111 : Determination of Minimum inhibitory concentration (MIC S) and minimum bactericidal concentration (MBC S) of the flowers of C.A. of different extracts

Methanol N-hexane Water The Bacterial Extract Extract Extract Strain tested MIC MBC MIC MBC MIC MBC A.A A.A A.A µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml Rhodococcuss equi 12.5 12.5 + 6.25 6.25 + - - - Staphylococcus aureus SARM 25 25 - 6.25 6.25 - - - - Staphylococcus aureus MLD 12.5 12.5 - 12.5 12.5 + - - - Streptococcus agalactiae 25 25 ------Enterococcus faecalis 25 25 - 12.5 12.5 + - - - Pseudomonas aeruginosa 25 25 - 12.5 12.5 + - - - Pseudomonas cepacia 25 25 - 12.5 12.5 + - - - Proteus mirabilis 12.5 12.5 - 12.5 12.5 + - - - Morganella morganii 25 25 - 12.5 12.5 + - - - + : Bactericidal , - : Bacteriostatic , A.A : Antibacterial activity .

Figure II. Determination of Minimum inhibitory concentration ( MIC S ) and Minimum bactericidal concentration (MBCS ) of the flowers of C.A of different extracts

Determination of Minimum inhibitory concentration (MICS) and

minimum bactericidal concentration (MBCS) of the flowers of C.A. of different extracts.

30

25 Methanol Extract MIC 20 Methanol Extract MBC 15

N-hexane Extract MIC value 10 N-hexane Extract MBC 5

0

P roteus

m irabilis m organii equi M organella faecalis

c epac ia agalac tiae aeruginosa Enterococcus aureus M LD Pseudom onas Pseudom onas Streptococcus Rhodococcuss aureus SARM Staphylococcus Staphylococcus

Gram - Gram +

The Bacterial Strain tested

According to the values of MIC and MBC, the methanol extract which is Bacteriostatic shows an MIC between 25 µg/ml and 12.5 µg/ml for sensitive strains, with hexane extract 12.5 µg/ml and 6.25 µg/ml was obtained, this extract

160 Abdul-Malik Abudunia et al J. Chem. Pharm. Res., 2014, 6(8):156-161 ______is bactericidal. Thus the hexane extract is more inhibitory than the methanol extract.

Thus, in our work we highlighted the antibacterial effect of hexane, methanol and aqueous extracts, Ours Identical results were obtained with Staphylococcus aureus , Escherichia coli, and Pseudomonas aeruginosa [27]. Similar results were obtained by Stayeh et al, 1998; bacterial inhibition was evaluated at 90% of bacterial strain [28]. in summary, we note that the extracts of the C.A. flowers has a clearly significant antibacterial action,

CONCLUSION

The results of the present study indicate that the methanol and n-hexane extracts of the C.A. flowers possesses a potential antibacterial activity .Further clinical studies are now required to determine the antibacterial principles of C.A. for various useful applications. The investigated plant extracts may be used for the preservation of processed foods as well as pharmaceutical and natural therapies for the treatment of infectious diseases in humans.

Acknowledgements The authors wish to thank Pr. M. Fanan of the Department of Botany , Scientific Institute of Rabat , for the botanical identification and collection of the plants used in this study.

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